Comparison of bromine and permanganate as ultrastructural stains for lignin in plants infected by the fungus Colletotrichum lagenarium.

Transmission electron microscopy (TEM) and energy dispersive X-ray microanalysis (EDS) were used to localize manganese from KMnO4, and bromine, as ultrastructural stains for lignin in an herbaceous plant. The Spookie cultivar of pumpkin is susceptible to infection by the fungus Colletotrichum lagenarium and served as a model system to compare the Br and KMnO4 techniques. Bromine was used in a fixation/staining procedure, and in separate experiments, KMnO4 was used as either a fixative or as a postsection stain. The technique for using bromine was modified from the woody plant procedure by adding a paraformaldehyde prefixation step. With the bromine procedure, cell walls were well-preserved, but the cytoplasm was heavily extracted. The KMnO4 procedures produced well-fixed cytoplasm, but with some staining artifacts. With all procedures, EDS dot mapping demonstrated lignin deposition in the cell walls specifically associated with sites of fungal infection. Lignin was also localized in secondary walls of tracheary elements, sites known to be highly lignified. The bromine procedure provided the most specific localization of lignin with a minimum of artifact. The specific applications of these stains provided data on the ultrastructural localization of lignin which contributed to the elucidation of its role in the interactions between pathogenic fungi in both their resistant and susceptible plant hosts.